West Nile Virus (WNV) is currently the most widely dispersed of all encephalitic flaviviruses, making it an important emerging disease worldwide. Infection with WNV produces severe neurological disease in approximately 1 percent of individuals. Little is known about the genetic determinants that have contributed to the dramatic dissemination of WNV to new geographic and host ranges and its neurovirulent phenotype. Genetic determinants for WNV adaption, fitness and neurovirulence will be investigated by inserting single point mutations in WNV infectious clones and evaluating the resulting phenotypes in birds, mosquitoes and mice. Specifically, I will (1) assess the phenotypic effects of polymorphisms at the NS3-249 site in different lineage 1 and 2 WNV backbones for modulation of avian host competence, (2) assess the neuroinvasive and neurovirulence effects of the NS3-249 mutation in different lineage 1 and 2 backbones in a mouse model, and (3) characterize the fitness and transmission potential of WNV lineage 1 and 2 viruses containing a NS3-249 mutations in enzootic and epizootic mosquito vectors.